Method for differentiating microorganisms in a sample
Culture media for microorganisms containing blood or hemin, particularly Trypticase Soy Agar with blood, and chocolate agar, are combined with known chromogenic substrates to produce chromogenic media. Methods for preparing these chromogenic media include adding chromogenic substrates to the surface of previously prepared media, or incorporating the chromogenic substrate into the media as it is prepared. Methods for distinguishing microorganisms in a sample using these culture media are also described.
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Differentiation and identification of microorganisms such as bacteria, fungi or yeasts in a sample suspected of containing pathogenic organisms is important. The non-pathogenic organisms must be distinguished from the pathogens. Most frequently, samples contain mixed bacteria, mixed fungi or mixed yeasts, whether a clinically obtained sample, or an environmental sample, e.g. from a water supply, or a food based sample, including poultry and dairy products.
While numerous methods have evolved to help distinguish and identify microorganisms, most involve appropriate selection of culture media and several additional steps including incubating, preliminary identification by morphology, followed by further culturing and incubation of selected microorganisms. In some instances, complete identification of the microorganisms in a sample can take several days, as the cultures are typically incubated for 18-24 hours before significant growth is detected.
By way of example, by formulating the culture media to permit the growth of only bacteria with certain characteristics, the growth of some bacteria (which are not of interest) can be suppressed. However, regardless of how culture media are adjusted, by altering ingredients or the concentrations of ingredients, selection of the culture media still may not typically result in clear identification and differentiation of all the bacterial species present in the sample. Microscopic examination of morphology and further testing are commonly required, or use of multiple, differing culture media.
Use of color to differentiate various microorganisms was attempted against this backdrop.
While use of color to differentiate and identify microorganisms has been known for many years, until recently, these methods suffered from many drawbacks, including deterioration of the color change, diffusion or evaporation of the color formed, generation of color which cannot distinguish between bacteria or which is difficult to read against the color of the media, or the necessity for several steps, including UV radiation to develop the color.
Relatively recently, so-called chromogenic media have been utilized to differentiate microorganisms.
The primary method employed by chromogenic media to detect and differentiate microorganisms is the reaction of glycosidase substrates, as shown below. This basic chemical reaction to form color is exemplified by X-Gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside), a reagent commonly used in molecular biology to monitor lacZ gene exprssion. This colorless substrate undergoes a reaction with galactosidase to produce an insoluble blue indigo product: ##STR1##
The oxidation step occurs rapidly to produce the indigo compound from the unstable 3-hydroxy indole. This oxidation may be air mediated and may also involve cellular metabolic processes in some organisms. One advantage of the indigo compounds as microbial indicators is their localization within the microorganisms, which helps to prevent deterioration and diffusion of the color change. The indigo compounds are also relatively nontoxic to growing microorganisms. Another advantage is that several colors are available by changing substituents on the indolyl ring. This allows differentiation of microorganisms with different glycosidase activities. A list of such colored substrate products is given below. ##STR2##
Products of known indolyl glycoside substrates:
______________________________________
R.sub.1 R.sub.2 R.sub.3 R.sub.4
Color
______________________________________
H H H H blue
Cl Br H H blue
H Br Cl H magenta
H H Cl H salmon
H I H H purple
H Br H H blue
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In addition to glycosidase substrates, dyes such as pH indicators are occasionally used in chromogenic media. Chromogenic indolyl substrates for esterases, phosphatases, and other enzymes are also used in some applications. These work similarly to the glycosidase substrates. An example, "Mag-Phos" is shown below. ##STR3##
Recently, many examples of testing methods utilizing this basic chemistry have been reported, some of which are described below.
Rapid identification of Salmonella is an important public health issue. Culture media for this purpose comprising a chromogenic compound linked to a C.sub.7 -C.sub.10 fatty acid, and an appropriate detergent which promotes liberation of the chromogenic compounds is proposed in WO94/09152. The chromogenic compound is preferably 5-bromo-4-chloro-3-indolyl caprylate, and the detergent is selected from fused polycyclic detergents. .beta.-glucosides and/or .beta.-galactosides are advantageously added.
Chromogenic compounds derived from indolylglucuronic acid as a substrate for the GUS enzyme are described in WO 94/08043.
Combining a chromogenic .beta.-galactosidase and a .beta.-galactosidase and a .beta.-glucoronidase in a test medium to distinguish between Escherichia coli and general coliforms in a single test with a single sample is disclosed in U.S. Pat. No 5,210,022.
A test medium useful for identifying bacteria found in urine samples containing a chromogenic .beta.-glucoronidase substrate capable of forming a first color when reacted with .beta.-glucoronidase, a chromogenic arylsulfatase substrate capable of forming a second color when reacted with arylsulfatase, and a nutrient base is described in U.S. Pat. No. 5,464,755. Proteinaceous opaque compounds, such as milk-derived compounds can be included in the test medium. A medium for isolating Salmonella colonies without ambiguity by use of specific coloration is disclosed in U.S. Pat. No. 5,194,374. This medium contains peptones, a polyol metabolizable by Salmonella and a pH indicator sensitive to acidification. The polyol is adsorbed on a pulverulent material. The medium may also contain deoxycholate and a chromogenic .beta.-galactosidase substrate.
A method for revealing the presence or absence of a particular microorganism strain, with at least one strain-specific enzyme substrate chromogen and at least one compound selected from a high concentration carbohydrate, are added to the culturing medium as disclosed in WO95/04156. Once the chromogen has been hydrolyzed, a color differing from the basic color of the chromophore results.
A method of identifying E. coli using a growth medium for E. coli with 8-hydroquinoline-.beta.-D-glucuronide as an activator such as X-glucuronide is disclosed in EP 0025467. This method shows the E. coli as darkly pigmented blobs.
Determining the presence of E. coli in a liquid sample passed through a suitable membrane filter by contacting the filter with a chromogenic reagent, indoxyl-.beta.-D-Glucuronide, in an E. coli nutrient medium followed by incubation is described in U.S. Pat. No. 4,923,804. The E. coli appears as an indigo blue color.
A method for identifying Enterobacteriaceae in a single culture medium is disclosed in U.S. Pat. No. 3,870,601. The media comprises a mixture of chromogenic .beta.-galactoside substrates with a decarboxylase substrate, a deaminase substrate, a urease subtrate, a hydrogen disulfide detection system, or a carbohydrate fermentation system. ONPG (o-nitrophenyl-.beta.-galactopyranoside) is disclosed as suitable.
Use of two different chromogens, and biological material capable of fermenting a sugar in a test medium adjusted to a pH conducive for color change of a pH indicator upon acidification upon fermentation of the sugar, is used to distinguish bacteria in WO 97/36001.
Direct detection of Salmonella (except S. arizonae) is possible by combining glucuronic acid and a pH indicator in the culture medium, together with a chromogenic or fluorogenic compound capable of being hydrolyzed by .beta.-galactosidase, as described in U.S. Pat. No. 5,434,056.
WO96/40861, like other art, is concerned with the identification of pathogens such as the possibly fatal E.coli 0157:H7 and Salmonella. A medium, liquid or solid, containing propionic acid, one or more chromogenic substrates, such as galactosidase substrates and glucuronidase substrates, and a nutrient base can identify these bacteria, according to this document.
Differentiation of pathogenic monocytogenes species of Listeria from the non-pathogens by use of a glycine amino peptidase substrate is disclosed in U.S. Pat. No. 5,330,889. Identification and differentiation of different species of yeasts can be accomplished using CHROMagar Candida plates available from CHROMagar Company, Paris, France. Yeasts from clinical samples grown on these plates are identified by variant colors and morphology. See e.g. A. P. Koehler, et al. J. Clin. Microbiol., 37, pp. 422-26 (1999). The CHROMagar medium is composed of 10 g peptone, 20 g glucose, 15 g agar, 0.5 g chloramphenicol, per liter, and a "chromogenic mixture," whose components are maintained in secrecy by the manufacturer. (E. T. S. Houang, et al., J. Clin. Path., 50, pp. 563-565 (1997).) The yeast colonies appear in colors such as pink, blue, apple green and rose on a clear background.
CHROMagar Salmonella (CAS) is used to identify Salmonella spp. as mauve colonies after 18 hours of incubation, while other members of the Enterobacteriaceae grow as blue or uncolored colonies. (O. Gaillot, et al., J. Clin. Microbiol., 37, pp. 762-65 (1999).)
While there are obviously many methods known and described for differentiating microorganisms, those currently known involve the use of culture media which are clear, thus providing good contrast and ready visibility when a color change takes place. Use of opaque media have also been used with chromogens, such as resulting from the addition of proteinaceous materials such as milk. Here again, however, the color change of susceptible bacteria is easy to detect.
Not all clinically important bacteria, yeasts or fungi can be cultured on the chromogenic media described above. For example, the so-called fastidious bacteria have specific growth requirements, and will not grow, (or grow in a meaningful way) on routine media. Examples of clinically important fastidious bacteria include Neisseria and Haemophilus. Many of these bacteria are found in respiratory, cerebral-spinal and genital fluids and secretions.
For these fastidious bacteria, incubation and growth is normally performed on chocolate agar, a culture medium containing hemoglobin. (See, Martin et al., Publ. Health Rep., 82:361 (1967). As its name implies, chocolate agar looks like chocolate and is brown in color. Therefore, whether chromogenic media will provide any meaningful contrast to permit differentiation and identification of bacteria is unknown.
Trypticase Soy Agar (TSA) with sheep blood is a culture media which is commonly used for the cultivation and isolation of fastidious microorganisms when distinct hemolytic reactions are important (e.g., Streptococcus pneumomiae from respiratory specimens). This culture media supports growth of many kinds of bacteria, which can only be distinguished by their morphology, which may be extremely difficult. Due to the presence of the blood, this media has a bright red color, and therefore it is unknown whether a color change would be discernible on this media.
The TSA with sheep blood media differentiates microorganisms on the basis of hemolysis, which results in the clearing (or lysis) of red blood cells due to the production of hemolysins by the microorganisms.
Due to their dark color it was unknown whether chromogenic reactions would be discernable on TSA and blood or chocolate agar plates. Moreover, it was unknown whether the presence of chromogens in the media would interfere with the hemolytic reaction on TSA with sheep blood media.
It is an object of this invention to prepare a chromogenic indicator medium containing blood or hemin for growing and identifying microorganisms.
It is another object of the present invention to prepare a TSA and sheep blood culture medium containing chromogenic substrates.
It is also an object of the present invention to differentiate microorganisms based on a change in color of the microorganisms on a TSA and sheep blood culture medium.
Another object of the present invention is to prepare a chocolate agar culture medium containing chromogenic substrates.
Yet another object of this invention is to differentiate bacteria based on a change in color of the bacteria on chocolate agar media.
SUMMARY OF THE INVENTIONThe present invention relates to a method for differentiating microorganisms in a sample utilizing a chromogenic indicator medium containing blood or hemin for growing and identifying microorganisms.
The present invention also encompasses a method for utilizing a chromogenic indicator media for color differentiating microorganisms, including bacteria, yeasts and fungi, in a culture medium containing blood or hemin such as Trypticase Soy Agar with 5% Sheep Blood (TSASB), or Chocolate agar. Other dye or color producing compounds, such as crystal violet, could be used in the media as well. The resulting chromogenic reactions are visible and easily readable on these non-clear, i.e., colored growth media. Surprisingly, the presence of the chromogens do not adversely affect the other differential properties of the growth media, such as hemolytic reactions, colony size or shape or other characteristics. This invention can be used for clinical and industrial (e.g. food, water, environmental or pharmaceutical) specimens, and includes a method for differentiating bacteria on TSASB and Chocolate agar growth media with chromogens. The invention also includes methods for preparing chromogenic indicator media containing blood or hemin.
DETAILED DESCRIPTION OF THE INVENTIONThe present invention uses chromogens in conjunction with known growth media TSASB and chocolate agar.
TSASB is commercially available. For example, pre-made plates containing this growth media can be obtained from Becton Dickinson Biosciences, Cockeysville, Md., under the name TSA II.TM.. Such plates are normally stable, under refrigeration, for 14-16 weeks.
Alternatively, TSASB can be made in according to known formulas and procedures. See, e.g. Dilworth, et al., J. Clin. Microbiol., 2:453 (1975).
Chocolate agar growth media are also commercially available, pre-plated and in tubed slants, from manufacturers such as Becton Dickinson Biosciences, Cockeysville, Md., under the name Chocolate Agar II.
Chocolate agar can also be made according to known formulas and procedures. See, e.g. Martin et al., Publ. Health Rep., 82:361 (1967).
As discussed in the Background herein, suitable chromogenic substrates are compounds which will be acted upon by enzymes found in the target bacteria to result in an insoluble colored product which is contained within the bacteria. Many such chromogenic substrates are known and each is targeted to a specific enzyme.
Examples of suitable chromogenic substrates are provided below. This listing is not meant to be all-inclusive. Other such compounds are known, and are available from companies such as Sigma (St. Louis, Ill.), INALCO (Milan, ITALY), BIOSYNTH (AG Staad, SWITZERLAND), BIOSYNTH INTERNATIONAL (Naperville, Ill.) and GLYCOSYNTH (Warrington, Cheshire, England).
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Abbreviation Chemical Name
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X-Acglmn 5-Br-4-Cl-3-indolyl-N-
Acetyl-.beta.-D-galactosaminide
X-Cap 5-Br-4-Cl-3-indolyl-
caprylate
BG-Gal 4-Cl-3-indolyl-.beta.-D-
galactopyranoside
Sal-Gal 6-Cl-3-indolyl-.beta.-D-
galactopyranoside
X-Gal 5-Br-4-Cl-3-indolyl-.beta.-D-
galactopyranoside
X-Glucoside 5-Br-4-Cl-3-indolyl-.beta.-D-
glucopyranoside
Sal-Glucuro 6-Cl-3-indolyl-.beta.-D-
glucuronide (CHA salt)
X-Gluc 5-Br-4-Cl-3-indolyl-.beta.-D-
glucuronide (CHA salt)
X-Glucuro (c) 5-Br-4-Cl-3-indolyl-.beta.-D-
glucuronide (CHA salt)
X-Glucuro (s) 5-Br-4-Cl-3-indolyl-.beta.-D-
glucuronide (sodium salt)
IPTG Isopropyl-.beta.-D-
thiogalactopyranoside
Mag-Phos 5-Br-6-Cl-3-indolyl-
phosphate, p-touidine salt
X-Sulfate 5-Br-4-Cl-3-indolyl-sulfate
(Potassium salt)
X-.alpha.-Glucoside
5-Br-4-Cl-3-indolyl-.alpha.-D-
glucopyranoside
X-Cello 5-Br-4-Cl-3-indolyl-.beta.-D-
cellobioside
X-Acetate 5-Br-4-Cl-3-indolyl-3-
______________________________________
______________________________________
Abbreviation Chemical Name
______________________________________
acetate
X-Glucosamine 5-Br-4-Cl-3-indolyl-.beta.-D-
glucosaminide
X-Butyrate 5-Br-4-Cl-3-indolyl-
butyrate
X-Palminate 5-Br-4-Cl-3-indolyl-
palminate
X-Phosphate 5-Br-4-Cl-3-indolyl-
phosphate
X-Fucoside 5-Br-4-Cl-3-indolyl-.beta.-D-
fucoside
X-Xylo 5-Br-4-Cl-3-indolyl-.beta.-D-
xylopyranoside
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Chromogenic substrates, such as those noted above, can be added to the growth media in at least two ways.
A small amount of the chromogenic substrate, from 0.05 to 0.2 g, preferably 0.05 to 0.1 g (e.g. 0.08 g) can be added to a small amount of DMSO (e.g. 1 ml.). Then a small aliquot of this solution (e.g. about 50 microliters) can be added to the surface of a pre-plated medium, either TSASB or chocolate agar, then distributed using a spreader. The plate is then allowed to dry 3-4 hours in a hood. It will be noted that in some instances a powdery insoluble precipitate forms during spreading.
Another method of incorporating the chromogenic substrates into the growth media is to freshly prepare the media in accordance with known procedures. All chromogens can be added aseptically to the base post autoclaving at a preferred concentration of 0.1 g/l (a suitable range is 0.05 to 0.20 g/l ). Chromogens are either pre-dissolved in DMSO or added as powder, if water soluble. Chromogens can also be added prior to autoclaving.
Other methods for incorporating the chromogenic substrates will be apparent to those of ordinary skill in the art.
The fungi which may be used in the present invention are those known to one of ordinary skill in the art and include Aspergillus spp., Trichosporn spp. and Geotrichum spp. Likewise, yeasts which may be evaluated in the present invention are known to those of ordinary skill and include Candida spp. and Cryptoccus spp.
The bacteria to be grown and differentiated using the present invention are those which are known to be suitable for growth on blood or hemin containing media. As noted previously, fastidious bacteria are intended for the Chocolate agar medium. Examples include Haemophilus spp. and Neisseriae spp.
Sources of such bacteria include clinical samples, such as sputum, urine and blood and industrial sources, such as food, water, environmental or pharmaceutical samples.
TSASB suitable bacteria are also well known. Examples are Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae.
Samples of suitable bacteria for TSASB can be obtained from many sources, including clinical samples and industrial samples.
Of course, as is well known, specific, identified bacteria can be obtained from sources such as ATCC, the American Type Culture Collection in Manassas, Va.
Specific examples of the use of various chromogenic substrates on both TSASB and chocolate agar media are provided below. The specific experiments set forth below demonstrate that various bacteria respond differently to the presence of chromogenic substrates.
Some bacteria will develop a color, such as dark blue, green or pink, that will differentiate them, perhaps in combination with a characteristic morphology, from other bacteria in a sample. In some cases, a particular bacteria may remain colorless, while others of the same species do develop color, thus providing another basis to differentiate the bacteria. Yet another possibility is that a particular bacterium will develop a color different from those of other bacteria in a sample. This provides yet another means for the bacteria to be differentiated from other bacteria in a sample.
EXAMPLE 1In this series of experiments, 0.08 g of each chromogen indicated below was added to 1.0 ml DMSO and thoroughly mixed. All the chromogens appeared to be soluble, providing a clear and/or colorless solution, except for Mag-Phos, which appeared clear and light orange in color.
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Abbreviation Chemical Name
______________________________________
X-Acglmn 5-Br-4-Cl-3-indolyl-N-
Acetyl-.beta.-D-galactosaminide
X-Cap 5-Br-4-Cl-3-indolyl-
caprylate
BG-Gal 4-Cl-3-indolyl-.beta.-D-
galactopyranoside
Sal-Gal 6-Cl-3-indolyl-.beta.-D-
galactopyranoside
X-Gal 5-Br-4-Cl-3-indolyl-.beta.-D-
galactopyranoside
X-Glucoside 5-Br-4-Cl-3-indolyl-.beta.-D-
glucopyranoside
Sal-Glucuro 6-Cl-3-indolyl-.beta.-D-
glucuronide (CHA salt)
X-Glucuro (c) 5-Br-4-Cl-3-indolyl-.beta.-D-
glucuronide (CHA salt)
X-Glucuro (s) 5-Br-4-Cl-3-indolyl-.beta.-D-
glucuronide (sodium salt)
IPTG Isopropyl-.beta.-D-
thiogalactopyranoside
Mag-Phos 5-Br-6-Cl-3-indolyl-
phosphate, p-touidine salt
X-Sulfate 5-Br-4-Cl-3-indolyl-sulfate
(Potassium salt)
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50 .mu.l of each chromogen solution was added to the surface of each of the following pre-plated media (one chromogen solution per plate):
TSAII 5% Sheep Blood;
Chocolate II; and
TSA plain (no blood).
All pre-plated media were obtained from Becton-Dickinson Microbiology Systems, MD.
The solution was dispersed using a spreader and allowed to dry 3-4 hours in a hood. In some instances a powdery, insoluble precipitate formed during spreading, except for X-Glucuro(s) which remained clear. After 1 week, however, some precipitate was evident only on the X-Glucoside and X-Acglmn plates.
The bacterial test strains were adjusted to a 0.5 McFarland equivalent and diluted 1:10 in sterile saline. The plates were then inoculated using the standard streak plate method.
The following bacteria were evaluated: Escherichia coli 25922; Staphylococcus aureus 25923; S. epidermidis 12228; Streptococcus pneumoniae 6303; Group B Strep. 12386; Haemophilus influenzae 35540, 10211; Branhamella catarrhalis 25238; Neisseria meningitidis 13090; N. sicca 29193; N. gonorrhoeae 35201; and Gardnerella vaginalis 14019.
All plates were incubated at 35.degree. C. for 20-24 hours. The TSAII with Sheep blood and TSA II (plain) were incubated in air, while the chocolate agar was incubated in CO.sub.2 (5%).
After incubation, the plates were evaluated for growth by quadrant readable colonies.
The fastidious strains of bacteria were only tested on Chocolate II Agar. Results of these tests are presented below in Tables I and II.
TABLE I
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Evaluation of Microorganism Colony Color Formation Following Growth on
TSA II
With 5% Sheep Blood, TSA plain and Chocolate II Agar Supplemented With
Various Chromogenic Substrates
__________________________________________________________________________
TSA Plain
TSA II 5SB
Choc II
Test Strain
Substrate
Growth
Color
Growth
Color
Growth
Color
__________________________________________________________________________
S. epi
X-Acglmn
4+ WT 4+ WT 4+ WT
12228 X-Cap NG NA 1+ WT NG NA
X-Gal 1+ WT 4+ WT 4+ WT
X-Glucoside
4+ WT 4+ WT 4+ WT
X-Glucuro
4+ WT 4+ WT 4+ WT
Mag-phos
1+ purple
1+ purple
1+ purple
E. coli
X-Acglmn
4+ NC 4+ NC 4+ NC
25922 X-Cap 4+ NC 4+ NC 4+ NC
X-Gal 4+ blue 4+ blue 4+ blue
X-Glucoside
4+ NC 4 NC 4+ NC
X-Glucuro
4+ blue 4+ blue 4+ blue
Mag-Phos
4+ purple
4+ purple
4+ purple
S. aureus
X-Acglmn
4+ NC 4+ NC 4+ NC
25923 X-Cap 1+ turquoise
4+ turquoise
4+ turquoise
(mixed) mixed
X-Gal 4+ NC 4+ NC 4+ NC
X-Glucoside
4+ NC 4+ NC 4+ NC
X-Glucuro
4+ NC 4+ NC 4+ NC
Mag-Phos
1+ purple
3+ purple
2-3+
purple
S. pneu:
X-Acglmn
1+= NC 4+= blue 4+= NC
6303 X-Cap NG NA < NC NG NC
X-Gal = NC = light blue
= NC
X-Glucoside
< NC = NC = NC
X-Glucuro
= NC = NC = NC
Mag-Phos
NG NA < NC < NC
B. catt
X-Acglmn
= NC = NC = NC
25258 X-Cap NG NA < NC < NC
X-Gal NG NA = NC = NC
X-Glucoside
< NC = NC = NC
X-Glucuro
>> NC = NC = NC
Mag-phos
NG NA << NC NG NC
Group B
X-Acglmn
= NC = NC = NC
Strep 12386
X-Cap NG NA < NC << NC
X-Gal < NC < NC = NC
X-Glucoside
= blue = blue = blue
X-Glucuro
= blue = blue (a)
= blue
Mag-phos
< purple
< purple (a)
= purple
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Substrates on Chocolate II Agar
Test Strain
X-Acglmn
X-Cap
X-Gal
X-Glucoside
X-Glucuro
Mag-Phos
__________________________________________________________________________
H. influ.
= NC = NC = NC = NC = NC = purple
35540
N. gono.
= NC NG < NC < NC NG NG
35201
G. vag. 14019
= NC NG = NC < NC NG NG
H. influ.
= NC NG = NC = NG = NC = purple
10211
N. sicca.
= NC blue = NC = NC = NC = purple
29193
N. mening.
< NC NG = NC = NC = NC = NC
13090
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Key:
NC = no Color
a = not as hemolytic
NG = no growth
WT = white
NA = not applicable
= growth equivalent to control sample
< colony size/quadrant less
TABLE II
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Summary of Table I Results
Substrates Producing
Substrates Producing
Test Strains
Color Growth Inhibition
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E. coli 25922
X-Gal, X-Glucuro,
None
Mag-phos
S. aureus 25923
X-Cap, Mag-phos
None
S. epidermidis 12228
Mag-phos X-Cap
S. pneumoniae 6303
X-Acglmn (TSA II
X-Cap, Mag-phos
only)
Group B Strep. 12386
X-Glucoside, X-
Slight inhib. W/ X-
Glucuro, Mag-phos
Cap, X-Gal, Mag-phos
H. influenzae 35540,
Mag-phos None
10211
B. catarrhalis 25238
None X-Cap, Mag-Phos
N. meningitidis
None Slight inhib w/ X-
13090 Acglmn
N. sicca 29193
X-cap, Mag-phos
None
N. gonorrhoeae 35201
None X-Cap, X-Glucuro,
Mag-phos
G. vaginalis 14014
None X-Cap, X-Gal, X-
Glucuro, Mag-phos
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It was surprising to discover that chromogenic substrates dissolved in the organic solvent dimethylsulfoxide (DMSO), when applied to the surface of or incorporated within a highly colored medium (i.e., a medium containing blood or hemin) could support the growth of microorganisms and result in color differentiation of the microorganisms.
The Haemophilus influenzae, both strains, appear dark purple on chocolate agar with Mag-phos. This is clearly differentiated from other sputum normal flora which appeared non-colored.
Group B streptococcus appears blue with beta hemolysis on TSASB with X-Glucuro.
Neisseria sicca appears purple on Chocolate agar with Mag-phos. N. meningitidis appears colorless.
In conducting these experiments, it was found that X-Cap and Mag-phos were somewhat insoluble, forming a white precipitate, when placed on the plated media. These two compounds also proved to have the most inhibitory effect. It is possible the concentration used for these compounds was too high.
EXAMPLE 2Using the procedure of Example 1, the bacteria tabulated below were evaluated on TSA II 5% Sheep Blood and Chocolate II agar. In each instance, the chromogens were added to the surface of the prepared-plated media at 0.004 g/plate in 50 .mu.l DMSO. The following bacteria were evaluated: Branhamella catarrhalis; Clostridium jejuni; Clostridium perfringens; Escheria coli; Enterococcus faecalis, Peptostreptococcus spp.; Streptococcus agalactiae; Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus mitis; Streptococcus pneumoniae; Streptococcus pyogenes; and Streptococcus groups C, F and G.
The results of this experiment, designed to evaluate the effect of growth conditions (incubation) on colony color formation, are tabulated below:
TABLE III
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TSA II 5% Sheep Blood (chromogens added to surface at 0.004 g/plate in 50
.mu.l DMSO)
Reference
Incubation
Strain No. Conditions
X-Acglm
X-Cap
X-Gal
X-Glucoside
X-Glucuro
Mag-Phos
__________________________________________________________________________
B. catarrhalis
25258
24 h, CO.sub.2
NC NC NC NC NC NG
C. jejuni
33291
5 d, MA
NC NG NC NC NC NG
C. jejuni
33292
5 d, MA
NC NG NC NC NC NG
C. perfringens
13124
5 d, ANA
blue (l)
NG blue (m)
NC NC NG
E. coli
25922
24 h, air
NC NC blue (d)
NC blue (d)
purple (m)
E. coli
25922
5 d, ANA
NC NC blue (d)
NC blue (m)
purple (m)
E. coli
25922
5 d, MA
NC NC blue (d)
NC blue (m)
purple (m)
E. faecalis
29212
24 h, CO.sub.2
NT NT NT NT NC NT
Peptostrep. spp
27337
5 d, ANA
NC NG NC NC NC NG
S. agalactiae
4768 24 h, CO.sub.2
NT NT NT NT blue (l)
NT
S. agalactiae
6638 24 b, CO.sub.2
NT NT NT NT blue (m)
NT
S. agalactiae
12386
24 h, CO.sub.2
NC NC NC blue (d)
blue (d)
purple (m)
S. agalactiae
12386
24 h, CO.sub.2
NT NT NT NT blue (d)
NT
S. agalactiae
13813
24 h, CO.sub.2
NT NT NT NT blue (d)
NT
S. agalactiae
BD11586
24 h, CO.sub.2
NT NT NT NT NC NT
S. agalactiae
BD747
24 h, CO.sub.2
NT NT NT NT grey blue
NT
S. aureus
25923
24 h, air
NC blue (l)
NC NC NC purple (m)
S. epidermidis
12228
24 h, air
NC NC NC NC NC purple (m)
S. mitis
6249 24 h, CO.sub.2
blue (vl)
NT NT NT NT NT
S. pneumoniae
6303 24 h, CO.sub.2
blue (l)
NC blue (l)
NC NC NC
S. pyogenes
19615
24 h, CO.sub.2
NT NT NT NT NC NT
Strep Grp C
12388
24 h, CO.sub.2
NT NT NT NT blue (d)
NT
Strep Grp F
12392
24 h, CO.sub.2
NT NT NT NT NC NT
Strep Grp F
BD77 24 h, CO.sub.2
NT NT NT NT NC NT
Strep Grp G
12394
24 h, CO.sub.2
NT NT NT NT grey blue
NT
(l)
Strep Grp G
27961
24 h, CO.sub.2
NT NT NT NT blue (d)
NT
__________________________________________________________________________
Abbrev:
NC = No Color
24 h = 24 hours
NT = Not Tested
5 d = 5 days
NG = no growth
CO.sub.2 = 5% CO.sub.2
vl = very light
MA = microaerophilic; 5-12% CO.sub.2 and 5-15% O.sub.2
l = light
ANA = anaerobic
m = medium
d = dark
TABLE IV
__________________________________________________________________________
Chocolate II Agar (chromogens added to surface at 0.004 g/plate in 50
.mu. DMSO
Reference
Incubation
Strain No. Conditions
X-Acglm
X-Cap
X-Gal
X-Glucoside
X-Glucuro
Mag-Phos
__________________________________________________________________________
B. catarrhalis
25258
24 h, CO.sub.2
NC NC NC NC NC NG
E. coli
25922
24 h, air
NC NC blue (d)
NC blue (d)
purple (m)
E. faecalis
29212
24 h, CO.sub.2
NT NT NT NT NC NT
G. vaginalis
14019
24 h, CO.sub.2
NC NG NG NC NG NG
H. influenzae
10211
24 h, CO.sub.2
NC NC NC NC NC purple (m)
H. influenzae
35540
24 h, CO.sub.2
NC NC NC NC NC purple (m)
N. 35201
24 h, CO.sub.2
NC NG NC NC NG NG
gonnorhoeae
N. meningitidis
13090
24 h, CO.sub.2
NC NG NC NC NC NC (col
dark)
N. sicca
29193
24 h, CO.sub.2
NC blue (m)
NC NC NC purple (m)
S. agalactiae
4768 24 h, CO.sub.2
NT NT NT NT blue (l)
NT
S. agalactiae
6638 24 h, CO.sub.2
NT NT NT NT blue (m)
NT
S. agalactiae
12386
24 h, CO.sub.2
NC NC NC blue (d)
blue (d)
purple (m)
S. agalactiae
12386
24 h, CO.sub.2
NT NT NT NT blue (d)
NT
S. agalactiae
13813
24 h, CO.sub.2
NT NT NT NT blue (d)
NT
S. agalactiae
BD11586
24 h, CO.sub.2
NT NT NT NT blue (vl)
NT
S. agalactiae
BD747
24 h, CO.sub.2
NT NT NT NT blue (l)
NT
S. aureus
25923
24 h, air
NC blue (l)
NC NC NC purple (m)
S. epidermidis
12228
24 h, air
NC NG NC NC NC purple (m)
S. mitis
6249 24 h, CO.sub.2
NC NT NT NT NT NT
S. pneumoniae
6303 24 h, CO.sub.2
NC NG NC NC NC NC
S. pyogenes
19615
24 h, CO.sub.2
NT NT NT NT NC NT
Strep Grp C
12388
24 h, CO.sub.2
NT NT NT NT blue (d)
NT
Strep Grp F
12392
24 h, CO.sub.2
NT NT NT NT NC NT
Strep Grp F
BD77 24 h, CO.sub.2
NT NT NT NT NC NT
Strep Grp G
12394
24 h, CO.sub.2
NT NT NT NT blue (d)
NT
Strep Grp G
27961
24 h, CO.sub.2
NT NT NT NT blue (d)
NT
__________________________________________________________________________
Abbrev:
NC = No Color
CO.sub.2 = 5% CO.sub.2
NT = Not Tested
NG = no growth
vl = very light
l = light
m = medium
d = dark
As will be observed from a review of the data tabulated above, X-Acglmn and X-Gal may be useful to differentiate C. perfringens from other Clostridia spp. grown under microaerphilic or anaerobic conditions. Also, X-Gal, X-Glucurono and Mag-Phos may be used to differentiate E. coli grown under microaerophilic or anerobic conditions, in addition to ambient air conditions.
EXAMPLE 3In this series of experiments, the chromogenic substrates were added directly into the culture medium as it was being prepared and prior to distribution to plates.
Laboratory prepared media consisting of TSA II base and 5% sheep blood were prepared in accordance with the procedures set out in Manual of BBL.RTM. Products and Laboratory Procedures, 4th ed., D. A. Power and P. J. McCuen (eds.), 1988. All chromogens were added aseptically to the base post autoclaving at a concentration of 0.1 g/l of media. Chromogens were either added as a powder (if water soluble) or predissolved in DMSO. In one instance, chromogens were added before autoclaving.
The chromogenic substrates used are set forth below:
In construing the chart below:
X=5-bromo-4-chloro-3-indolyl
Mag=5-bromo-6-chloro-3-indolyl
Sal=6-chloro-3-indolyl
______________________________________
Chromogenic Substrate Abbr.
______________________________________
X-N-Acetyl-.beta.-D-galactosaminide
X-Acglmn
Magenta-caprylate M-Cap
X-galactopyranoside (X-gal) + IPTG
X-Gal + IPTG
X-galactopyranoside X-Gal
X-glucoside X-Glucoside
Sal-glucuronide Sal-Glucuro
Magenta-phosphate Mag-Phos
X-Sulfate X-Sulf
X-Glucuronide X-Glucuro
Mix (Sal-Glucuro, Mag-phos, X-Acglmn, BG-Gal)
Mix
TSA II 5% SB - no chromogens
TSA cont.
______________________________________
A total of 55 strains (abbreviations as defined in Table V) were tested using the above media. Plates were incubated in CO.sub.2 and air, or CO.sub.2 only. All plates were read at 18-24 h for color, hemolytic activity, and growth as compared to the non-chromogenic control.
Colors were evaluated using Pantone Equivalents in order to more precisely and consistently identify the resulting colors, as noted below
______________________________________
Color Pantone Equivalent
______________________________________
b = blue 17-4336 .fwdarw. 19-4340
gb = grey blue 17-4412
p = purple 18-3513
gp = grey purple 15-3807
pb = purple blue 18-3932
NG = no growth
______________________________________
Used in front of the color abbreviations noted above, d=dark; l=light; vl=very light.
The bacterial strains were prepared in normal saline at 0.5 McFarland and diluted 1 in 10.
The plates were inoculated using the standard streak plate method or using a Steers replicator. Steers, E. et al., Antibiot. Chemother., 9:307-311 (1959).
Results are tabulated below in Tables V and VI.
TABLE V
__________________________________________________________________________
Colony Color on Various Chromogenic TSA II with 5% Sheep Blood
(Summary)
No. Chromogens
Strain Abbrev.
Tested
X-Acglmn
X-Gal
X-Glucoside
X-Glucuro
Mag-phos
M-Cap
X-Sulf
__________________________________________________________________________
Aeromonas hydrophilia
aehy
3 + 0 v 0 + 0 0
Branhamella catarrhalis
brca
2 0 0 0 0 v 0 0
Candida albicans
caal
2 + 0 0 0 0 0 0
Citrobacter freundii
cifr
2 0 + 0 0 0 0 +
Entercoccus faecalis
enfa
6 + v + 0 + 0 0
E. faecium enfu
1 + + + 0 + 0 0
E. coli (non-0157)
esco
3 0 + 0 + v 0 0
Klebsiella pneumoniae
klpn
3 0 + + 0 v 0 0
Pseudomonas aeruginosa
psae
3 0 0 v 0 0 0 0
Salmonella spp.
sasp
2 0 0 0 0 + 0 0
Serratia marcescens
sema
1 + + + 0 0 0 0
Streptococcus agalactiae
stag
3 0 0 + 0 + 0 0
Staphylococcus aureus
stau
5 0 0 0 0 v 0 0
Staphylococcus Coag Neg
step
5 v v 0 0 v 0 0
S. milleri stmi
2 0, <grth
0 0 0 no growth
0 0
S. pneumoniae
stpn
6 0, <grth
0 v 0 0, 0 0
<growth
S pyogenes stpy
6 0, <grth
0 v v +, 0 0
<grth
55
__________________________________________________________________________
+ = color detected
v = color varied slightly with strain
0 = no color detected
<= decreased growth vs. control
TABLE VI
__________________________________________________________________________
Colony Color on Various Chromogenic TSA II with 5% Sheep Blood
(Detail)
Refer-
Incub
Incub X-Gluc-
X- Sal- X-
Strain
ence No.
atm
time
X-Acglm
M-Cap
Xgal/IPTG
X-Gal
oside
Glucuro
Glucuro
Mag-Phos
Sulf
Mix TSA
__________________________________________________________________________
control
aehy
7965 CO.sub.2
21 h
gb 0 g gb 0 NT 0 gp 0 pb l yellow
aehy
7966 CO.sub.2
21 h
gb 0 0 gb g NT 0 gp 0 pb yellow
aehy
49847
CO.sub.2
21 h
gb 0 lg gb gb NT 0 g 0 pb yellow
brca
25238
CO.sub.2
21 h
0 0 0 0 0 NT 0 0 0 0 grey
brca
25240
CO.sub.2
21 h
0 0 0 0 0 NT 0 p 0 0 l grey
caal
10231
CO.sub.2
24 h
gb 0 0 0 0 NT 0 0 0 gb grey
cakr
34135
CO.sub.2
21 h
0 0 0 0 0 NT 0 lgp 0 0 grey
cifr
8454 CO.sub.2
21 h
0 0 gb gb 0 NT 0 0 0 0 grey
cifr
33128
CO.sub.2
21 h
0 0 gb gb 0 NT 0 0 gb gp grey
enfa
597 CO.sub.2
21 h
db 0 0 0 b NT 0 lp 0 db grey
enfa
10741
CO.sub.2
21 h
db 0 lgb gb db NT lp 0 db grey
enfa
12953
CO.sub.2
21 h
db 0 0 0 db NT 0 lp 0 b grey
enfa
14506
CO.sub.2
21 h
db 0 0 lgb db NT 0 lp 0 db grey
enfa
29212
CO.sub.2
21 h
db 0 gb gb db NT 0 0 db grey
enfa
29212
CO.sub.2
24 h
db 0 lgb vlgb
b NT 0 p 0 db grey
enfu
49032
CO.sub.2
21 h
db 0 0 gb b NT 0 vlgp 0 vlgp
grey
esco
11775
CO.sub.2
21 h
0 0 b b 0 NT 0 lp 0 gp grey
esco
25922
CO.sub.2
24 h
0 0 b b 0 NT 0 lgb 0 b grey
esco
33605
CO.sub.2
21 h
0 0 gb gb 0 NT 0 0 0 gp grey
klpn
13883
CO.sub.2
21 h
0 0 gb gb gb NT 0 0 0 lp grey
(mucoid)
klpn
33495
CO.sub.2
24 h
0 0 b b b NT 0 p 0 b grey
klpn
33606
CO.sub.2
21 h
0 0 gb gb gb NT 0 0 0 lp grey
milu
9341 CO.sub.2
21 h
0 0 0 0 0 NT 0 NG 0 0 l yellow
psae
9027 CO.sub.2
21 h
0 0 0 0 0 NT 0 0 0 0 grey
psae
15442
CO.sub.2
21 h
0 0 0 0 0 NT 0 0 0 0 yellow
psae
33607
CO.sub.2
21 h
0 0 0 0 lgb NT 0 0 0 0 l yellow
saty
19430
CO.sub.2
21 h
0 0 0 0 0 NT 0 p 0 p grey
satym
14028
CO.sub.2
21 h
0 0 0 0 0 NT 0 p 0 p grey
sema
13880
CO.sub.2
21 h
gb 0 gb gb gb NT 0 0 0 gb grey
stag
4768 CO.sub.2
21 h
0 0 0 0 b NT 0 lp 0 lp grey
stag
12386
CO.sub.2
24 h
0 0 0 0 b NT gp lp 0 p white
stag
12386
CO.sub.2
21 h
0 0 0 0 b NT 0 p 0 p white (beta)
stau
25923
CO.sub.2
24 h
0 0 0 0 0 NT 0 lp 0 lp white
stau
1006 CO.sub.2
21 h
0 0 0 0 0 NT 0 0 0 0 white (beta)
stau
1007 CO.sub.2
21 h
0 0 0 0 0 NT 0 0 0 0 white (beta)
stau
1008 CO.sub.2
21 h
0 0 0 0 0 NT 0 vlp 0 vlp white
stau
1009 CO.sub.2
21 h
0 0 0 0 0 NT 0 vlp 0 vlp white (beta)
step
12228
CO.sub.2
21 h
0 0 0 0 0 NT 0 0 0 vlp white
step
cdc1 CO.sub.2
21 h
0 0 0 0 0 NT 0 lp 0 lp grey
step
cdc2 CO.sub.2
21 h
0 0 0 0 0 NT 0 0 0 0 white
step
cdc3 CO.sub.2
21 h
0 0 b b 0 NT 0 0 0 0 white
step
cdc4 CO.sub.2
21 h
0 0 0 0 0 NT 0 lp 0 0 grey
stmi
6249 CO.sub.2
21 h
NG 0 0 0 0 NT 0 NG NG NG NG
stpn
6303 CO.sub.2
24 h
0 0 0 0 0 NT 0 0 0 0 alpha
stpn
6305 CO.sub.2
24 h
0 0 0 0 0 NT 0 0 0 0 alpha
stpn
1259 CO.sub.2
21 h
0 0 0 0 0 NT 0 0 0 0 alpha
(mucoid)
stpn
1260 CO.sub.2
21 h
0 0 0 0 0 NT 0 0 0 NG alpha
stpn
1331 CO.sub.2
21 h
0 0 0 NG NG NT NG NG NG NG NG
stpn
1628 CO.sub.2
21 h
0 0 0 0 0 NT 0 0 0 0 alpha
stpy
19615
CO.sub.2
21 h
0 0 0 0 gb NT 0 lp 0 lp white (beta)
stpy
49117
CO.sub.2
21 h
0 0 0 0 0 NT 0 lp 0 lp white (beta)
stpy
51339
CO.sub.2
21 h
0 0 0 0 0 NT 0 lp 0 lp white (beta)
stpy
1027 CO.sub.2
21 h
0 0 0 0 0 NT 0 vlp 0 vlp NG
stpy
1028 CO.sub.2
21 h
0 0 0 0 lgb NT 0 vlp 0 vlp grey (beta)
stpy
1029 CO.sub.2
21 h
0 0 0 0 lgb NT 0 vlp 0 vlp NG
stpy
1030 CO.sub.2
21 h
0 0 0 0 gb NT 0 vlp 0 vlp grey (beta)
caal
10231
air
24 h
0 0 0 0 0 NT 0 0 0 0 grey
(darker)
enfa
597 air
21 h
b 0 0 0 b NT 0 lp 0 db grey
enfa
10741
air
21 h
b 0 lb lgb b NT 0 p 0 db grey
enfa
12953
air
21 h
b 0 0 0 b NT 0 p 0 db grey
enfa
14506
air
21 h
b 0 0 0 b NT 0 p 0 db grey
enfa
29212
air
21 h
b 0 lgb gb b NT 0 p 0 db grey
enfa
29212
air
24 h
db 0 lgb vlgb
b NT 0 p 0 db grey
enfu
49032
air
21 h
lb 0 0 db b NT 0 lgp 0 lp grey
(gb48h)
esco
25922
air
24 h
0 0 b b 0 NT 0 lgb 0 b grey
klpn
33495
air
24 h
0 0 b b b NT 0 p 0 b grey
stag
4768 air
21 h
0 0 0 0 lb NT 0 p 0 lp grey
stag
12386
air
24 h
0 0 0 0 b NT gp lp 0 p grey
stau
25923
air
24 h
0 0 0 0 0 NT 0 lp 0 lp white
step
12228
air
21 h
0 0 0 0 0 NT 0 lp 0 0 white
(p-48h)
step
cdc1 air
21 h
0 0 0 0 0 NT 0 lp 0 lp grey
step
cdc2 air
21 h
0 0 0 0 0 NT 0 0 0 0 white
step
cdc3 air
21 h
lb 0 b b 0 NT 0 0 0 0 white
step
cdc4 air
21 h
0 0 0 0 0 NT 0 lp 0 vlp grey
stmi
6249 air
21 h
0 0 0 0 0 NT 0 0 0 NG NG
stpn
6303 air
24 h
0 0 0 0 0 NT 0 db 0 0 alpha
stpn
6305 air
24 h
0 0 0 0 0 NT 0 0 0 0 alpha
stpy
19615
air
21 h
0 0 0 0 lgb NT 0 lp 0 lgb white (beta)
(p48h)
stpy
49117
air
21 h
0 0 0 0 0 NT 0 p 0 lp white (beta)
stpy
51339
air
21 h
0 0 0 0 lgb NT 0 p 0 lp white (beta)
aehy
7965 CO.sub.2
18 h
db NT NT NT 0 0 NT NT NT NT grey hem
aehy
7966 CO.sub.2
18 h
db NT NT NT 0 0 NT NT NT NT grey hem
aehy
49847
CO.sub.2
18 h
db NT NT NT gb 0 NT NT NT NT grygrn hem
brca
25238
CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT grey
brca
25240
CO.sub.2
18 h
NG NT NT NT 0 0 NT NT NT NT grey
caal
10231
CO.sub.2
18 h
b NT NT NT 0 0 NT NT NT NT grey
cakr
34135
CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT grey
cifr
8454 CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT dark grey
cifr
33128
CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT grey
enfa
597 CO.sub.2
18 h
db NT NT NT db 0 NT NT NT NT grey
enfa
10741
CO.sub.2
18 h
db NT NT NT db 0 NT NT NT NT grey
enfa
12953
CO.sub.2
18 h
db NT NT NT db 0 NT NT NT NT grey
enfa
14506
CO.sub.2
18 h
db NW NT NT db 0 NT NT NT NT grey
enfa
29212
CO.sub.2
18 h
db NT NT NT db 0 NT NT NT NT grey
enfu
49032
CO.sub.2
18 h
lb NT NT NT db 0 NT NT NT NT grey
esco
11775
CO.sub.2
18 h
0 NT NT NT 0 b NT NT NT NT grey
esco
33605
CO.sub.2
18 h
0 NT NT NT 0 b NT NT NT NT grey hem
klpn
13883
CO.sub.2
18 h
0 NT NT NT b 0 NT NT NT NT white grey
klpn
33495
CO.sub.2
18 h
vlgb NT NT NT gb 0 NT NT NT NT white grey
klpn
33606
CO.sub.2
18 h
0 NT NT NT gb 0 NT NT NT NT white grey
miiu
9341 CO.sub.2
18 h
ly NT NT NT ly ly NT NT NT NT lt yellow
psae
9027 CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT dark grey
psae
15442
CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT dark grey
psae
33607
CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT dark grey
saty
19430
CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT grey
satym
14028
CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT dark grey
sema
13880
CO.sub.2
18 h
gb NT NT NT bg 0 NT NT NT NT dark grey
stau
25923
CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT white
stau
t006 CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT white
stau
t007 CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT white
stau
t008 CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT white
stau
t009 CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT white
step
12228
CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT white
step
cdc1 CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT white
stha
cdc2 CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT white
stsa
cdc3 CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT white
step
cdc4 CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT white beta
stmi
6242 CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT alpha
stpn
t259 CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT alpha
stpn
t260 CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT alpha
stpn
t331 CO.sub.2
18 h
drkr<muc
NT NT NT 0 0 NT NT NT NT alpha muc
stpn
t628 CO.sub.2
18 h
0 NT NT NT bg 0 NT NT NT NT alpha
stpy
51339
CO.sub.2
18 h
0 NT NT NT 0 db beta
NT NT NT NT beta
stpy
t027 CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT beta
stpy
t028 CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT beta
stpy
t029 CO.sub.2
18 h
0 NT NT NT 0 0 NT NT NT NT beta
stpy
t030 CO.sub.2
18 h
0 NT NT NT lgb 0 NT Nt NT NT beta
__________________________________________________________________________
Strain Abbreviation as defined in Table V
NG = Growth
NT = Not tested
0 = No Color
alpha = alpha hemolysis
beta = beta hemolysis
Again, it was surprising to discover that chromogenic substrates applied to the surface of, or incorporated within a highly colored medium (i.e., a nutrient medium containing blood or hemin) could help to identify and/or differentiate microorganisms.
As will be observed from review of the data tabulated above, Group B Strep (3 of 3 strains) gave blue colonies on X-glucoside TSA II. Colony hemolytic activity was slightly reduced. The CAMP test works well on X-glucoside TSA II (as described in the Manual of BBL.RTM. Products and Laboratory Procedures). PYR test (as described in Manual of Clinical Microbiology, 6th ed., P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 1995 ASM Press, Washington, D.C.) would be needed to rule out S. pyogenes or Group D strep which may also be blue. 4 of 7 S. pyogenes gave light grey blue color. 7 of 7 Enterococcus gave a blue color.
Group D Strep (7 of 7 strains) gave dark blue colonies on X-Acglmn. A few Gram Negative strains, one C. albicans and one S. epidermidis also gave blue colonies.
CO.sub.2 incubations typically gave slightly stronger color reactions vs. air incubation for all chromogens.
Addition of the X-chromogens prior to autoclaving did not effect performance.
M-Cap and X-Sulf were inactive for most organisms. Literature suggests that sodium desoxycholate may be important for the M-Cap reaction with Salmonella spp. One strain of Citrobacter gave a grey blue with X-Sulf TSA II.
Some of the chromogens, especially magenta substrates, caused the blood agar to darken after incubation, particularly after CO.sub.2 incubation. This darkening of the plate reduced the readability of the purple color reactions and hemolytic reactions.
X-gal was not particularly useful for Gram-positives. IPTG appeared to reduce the reactivity for some strains with X-gal. E. faecium (ATCC 49032) gave dark blue on X-Gal but no color on X-Gal with IPTG.
As would be expected, chromogens that produced colony colors that clearly contrast with the red surface of the blood plate are optimal. The blue-chromogen, X-substrate (5-bromo-4-chloro-3-indoxyl) has an absorption max (nm) of 615 which is most differentiated from the red blood plates color of approx. 515 nm. Some chromogens which produce purple/red/magenta colonies are not as easy to read against the red blood plate background.
Based on the above data, the color contrast can be summarized as follows:
______________________________________
Absorption
Contrast on
Chromogen Abbr. Colony max (nm)
Blood Plate
______________________________________
5-Bromo-4-chloro-
X- blue 615 Good
3-indoxyl
5-Bromo-6-chloro-
Mag- purple/red
565 Fair/poor
3-indoxyl
6-chloro-3-indoxyl
Sal- grey purple
540 Poor*
______________________________________
*Compound unstable, gave poor reactions with positive control (E. coli
25922)
While the examples use TSASB and chocolate agar as the blood or hemin containing culture media, the invention is not limited to these two media.
It is expected that other, similar blood or hemin containing media, such as Columbia Agar Base, with the addition of blood, which is widely used in Europe, would also be useful in the present invention. This media is commercially available, and contains more amino acid and carbohydrate than TSASB.
It is noted, however, that limited efforts to use commercially available Brucella Agar with blood, produced bacterial growth but no color change.
The above Examples are intended to be purely exemplary, and are not intended to in any way limit the scope of the present invention.
Claims
1. A method for differentiating microorganisms in a sample comprising:
- (a) growing said microorganisms on a chromogenic indicator medium which comprises:
- (i) a nutrient containing blood or hemin; and
- (ii) a chromogen; and
- (b) detecting a color change among the microorganisms grown.
2. The method according to claim 1 wherein component (i) of said chromogenic indicator medium is Trypticase Soy Agar and Blood.
3. The method according to claim 1 wherein component (i) of said chromogenic indicator medium is chocolate agar.
4. The method according to claim 1 wherein said microorganisms are bacteria.
5. The method according to claim 1 wherein said microorganisms are yeasts.
6. The method according to claim 1 wherein the microorganisms are obtained from a clinical source.
7. The method according to claim 1 wherein the microorganisms are obtained from an industrial source.
8. The method according to claim 1 wherein the microorganisms are obtained from a food source.
9. The method according to claim 1 wherein the chromogen is an X-linked chromogen.
10. The method according to claim 1 wherein the chromogen is a Magenta-linked chromogen.
11. The method according to claim 1 wherein the chromogen is a fluorogenic chromogen.
12. The method according to claim 1 wherein the chromogen has an absorption maximum of between about 400 nm and 800 nm.
13. The method according to claim 11 wherein the chromogen has an absorption maximum of between about 10 nm and 400 nm.
14. The method according to claim 3 wherein the microorganisms are bacteria.
15. The method according to claim 14 wherein the bacteria are obtained from a clinical source.
16. The method according to claim 14 wherein the bacteria are obtained from an industrial source.
17. The method according to claim 14 wherein the bacteria are obtained from a food source.
18. The method according to claim 14 wherein the chromogen is an X-linked chromogen.
19. The method according to claim 14 wherein the chromogen is a Magenta-linked chromogen.
20. The method according to claim 14 wherein the chromogen is a fluorogenic chromogen.
21. The method according to claim 14 wherein the chromogen has an absorption maximum of between about 400 nm and 800 nm.
22. The method according to claim 20 wherein the chromogen has an absorption maximum of between 10 nm and 400 nm.
23. A method for differentiating microorganisms in a sample comprising: a) applying a sample suspected of containing microorganisms of interest to a chromogenic indicator medium comprising (i) a nutrient containing blood or hemin; and (ii) a chromogen; b) incubating the sample for a period sufficient to permit detectable growth; and c) determining the visible presence or absence of microorganisms of interest.
24. The method according to claim 23 wherein component (i) of said chromogenic indicator medium is Trypticase Soy Agar and Blood.
25. The method according to claim 23 wherein component (i) of said chromogenic indicator medium is chocolate agar.
26. The method according to claim 23 wherein said microorganisms are bacteria.
27. The method according to claim 26 wherein said bacteria is Haemophilus influenzae.
28. The method according to claim 23 wherein said microorganisms are yeasts.
29. The method according to claim 23 wherein the microorganisms are obtained from a clinical source.
30. The method according to claim 23 wherein the microorganisms are obtained from an industrial source.
31. The method according to claim 23 wherein the microorganisms are obtained from a food source.
32. The method according to claim 23 wherein the chromogen is an X-linked chromogen.
33. The method according to claim 23 wherein the chromogen is a Magenta-linked chromogen.
34. The method according to claim 33 wherein said Magenta-linked chromogen is Magenta-phosphate.
35. The method according to claim 23 wherein the chromogen is a fluorogenic chromogen.
36. The method according to claim 23 wherein the chromogen has an absorption maximum of between about 400 nm and 800 nm.
37. The method according to claim 35 wherein the chromogen has an absorption maximum of between 10 nm and 400 nm.
38. A method of preparing chromogenic media containing blood or hemin comprising applying a chromogenic substrate to a surface of a previously prepared nutrient media, wherein the chromogenic substrate is carried in a solvent.
39. A method of preparing chromogenic media containing blood or hemin comprising adding a chromogenic substrate to a culture medium when the culture medium is prepared and prior to distribution to plates or tubes.
- Ashdown, L.. R.; "Pathology"; vol. 11(2), pp. 293-297, Feb. 1979. Wu, William, G.; "Medical Microbiology"; 2nd Edition, pp. 349, 1989.
Type: Grant
Filed: Sep 28, 1999
Date of Patent: Oct 10, 2000
Assignee: Becton, Dickinson and Company (Franklin Lakes, NJ)
Inventors: C. Michael Gosnell (Fallston, MD), Carrie A. Hughes (Parkton, MD), Paul E. Goldenbaum (Hampstead, MD)
Primary Examiner: Louise N Leary
Attorney: Bruce S. Weintraub, Esq.
Application Number: 9/407,638
International Classification: C12Q 118; C12Q 104; C12Q 102;
